In Situ Inhibitor Synthesis and Screening by Fluorescence Polarization: An Efficient Approach for Accelerating Drug Discovery

Abstract Target‐directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high‐throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl‐hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone‐based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.

. Reticulocyte evaluation of 17 in vivo [3] . Reticulocytes and reticulocyte % were measured 72 h (three times p.o. administration) after treatment with representative compounds (20 mg· kg -1 ). Errors: mean ± SEM. The results indicate that compound 17 exhibits a similar reticulocyte effect as does Roxadustat. P values were analyzed by the one-way ANOVA test comparing with the vehicle group (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Figure S10. Acute oral toxicity of tests for compound 17. The effects on body weight (A/B), and organ/body weight ratio (C/D) in mice (400 mg· kg -1 ). Blood biochemistry analysis of the indicators (E), including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) in mice, errors: mean ± SEM. The acute toxicity studies revealed no obvious abnormality after treatment with 17 (LD50 >400 mg· kg -1 ). There were no significant differences in the body weight, organ/body weight ratio, and blood biochemistry indexes (ALT, AST, BUN, and CRE) between the 17-treated group and the vehicle group. Figure S11. Subacute oral toxicity of compound 17. The effects of compound 17 on body weight (A/B), and organ/body weight ratio (C/D) in mice (po, 15, 50, and 100 mg· kg -1 ). (E-H) Blood biochemistry analysis of the indexes of BUN ALT, AST, and CRE in ICR mice. (I) H&E staining of major organs from ICR mice after treatment of 0.5% CMCNa and 17 (100 mg· kg -1 ) on alternate days for two weeks, error: mean ± SEM. The subacute oral toxicity study (14 days) did not exhibit any treatment-associated adverse effects on behavior, body weight, organ/body weight ratio, and blood biochemistry indexes (BUN ALT, AST, and CRE) at doses of 15, 50, and 100 mg· kg -1 . The major organs were further investigated using hematoxylin and eosin (H&E) staining. The 17-treated group (100 mg· kg -1 ) exhibited normal architecture comparable to the vehicle. Scale bar: 50 μm.

In-vitro evaluation
2.1 Fluorescence polarization (FP) competition assay of PHD2 [4] FP assays were performed using a SPARK Multi-Mode Microplate Reader (Tecan) with excitation and emission filters appropriate The reaction mixtures were incubated at RT for 60 min, then total fluorescence and fluorescence polarization measurements were taken. The percentage inhibition was calculated using Equation 1, where mPfree is the signal for the free probe (blank control) and mPbound is the signal for the bound probe (negative control).
IC50s were determined for duplicate measurements by non-linear least-squares analysis using GraphPad Prism 8.0.
The Ki values of the inhibitors were calculated using the Cheng-Prusoff equation. [5] Cheng-Prusoff equation: The Kd value was determined using a constant concentration of probe and titrating with PHD2 protein at increasing concentrations.
[L] represents the concentration of the PHD2 protein used in the FP assay.

Molecular Modeling
A structure of PHD2 in complex with Fe and a 2OG competing inhibitor (PDB ID: 4KBZ) was obtained from PDB website. The structure files of protein and compounds for docking were prepared using the Discovery Studio (DS) 2020 software. A water molecule HOH624, conserved in the active binding site of PHD2, was retained during the docking. Residues of PHD2 around the native ligand (radius = 10 Å) were defined as the binding sites for docking. Subsequently, the ligand was removed; the Fe (II) was retained during the docking. Compounds were docked into the binding sites using GOLD 5.1. Docking procedures were performed using the default setting with 100 genetic algorithm (GA) runs of ligands. For each GA run, a maximum of 125,000 operations was performed. When the top ten solutions possessed RMSD values within 1.5 Å, the docking was terminated. These obtained small molecule-protein complexes were visualized using PyMOL (TM) Molecular Graphic System, Version 2.3.0. [8] 17 was preincubated with microsomes from different species (0.5 mg/mL) at 1 μM for 5 min at 37 °C in 100 mM PBS buffer (pH 7.4). Subsequently, the bioreaction was catalyzed by adding NADPH (1 mM). After co-incubation for different times (0, 15, 30, 45, and 60 min) at 37 °C, the bioreaction was terminated by addition of cold acetonitrile. The clear supernatant samples were investigated by LC-MS/MS. Compound 17 (100 μM) was incubated with rat plasma at different times. The samples were centrifuged at 5000 rpm for 10 min; the supernatants were then analyzed by HPLC.

In vivo reticulocytes increased assay
Representative compounds and Roxadustat were formulated in normal saline solution. Mice (C57BL/6J, n=5 per dose level) were dosed p.o. with a volume of 0.2 mL. After three consecutive days of administration, these test samples were obtained via orbital venous plexus. Subsequently, these samples were analyzed for reticulocytes in the center for new drug safety evaluation and research of China Pharmaceutical University (ADVIA 2120 hematology system).

In vivo EPO increased assay
Plasma EPO was evaluated using the erythropoietin quantikine ELISA kit for mice (MEP00B). 17 and Roxadustat were formulated in normal saline solution. C57BL/6J (n=5) mouse were dosed p.o. in 0.2 mL (10, 20, 50 mg· kg -1 ). After 4 hours, these test samples were obtained via orbital venous plexus. Plasma was collected by centrifugation at 3000 rpm, these plasmas were detected by EPO ELISA.

Improvement of anemia induced by cisplatin administration
C57BL/6J mice were treated with cisplatin (po, 7 mg· kg -1 ) on days 0, 7, 14, and 28 of the experiment. On the 29th day, the animals were randomized into sham, control, and treated groups (n=5). Then, 17 and Roxadustat (5, 10, 20 mg· kg -1 ) were administrated for the treatment anemia caused by cisplatin. The mice were treated with the compounds every alternate day for 30 days. the test sample is obtained via orbital venous plexus. The hemoglobin of samples was analyzed in the center for new drug safety evaluation and research at China Pharmaceutical University (ADVIA 2120 hematology system). SUPPORTING INFORMATION S16 6. Subacute and acute oral toxicity assay [10] The experimental mice (SPF, ICR, male and female) were obtained from Nantong University (permit number: SCXK (Su) 2019-0001). All animal related experiments were conducted under China Pharmaceutical University guidelines (certification of using the housing facility for laboratory animals: SYXK (Su) 2018-0019) IACUC approved protocols in compliance with the guide for the care and use of laboratory animals (number: 202013705).
6.1 Subacute oral toxicity assay ICR mice were divided into 8 groups (n = 6), including 15 mg· kg -1 , 50 mg· kg -1 , 100 mg· kg -1 and vehicle groups for males and females, respectively. Experimental mice were treated with 17 through oral administration every other day for 14 days. The body weight of the mice was recorded. The mice were then sacrificed and dissected; the heart, kidney, liver, spleen, lung were extracted. Further, the blood was collected. Blood biochemistry assays were carried out as follows: The blood samples were centrifuged at 5000 rpm for 20 min, and the supernatant was subjected to blood biochemistry analysis. Typical functional indicators including urea; CRE, creatinine; AST, aspartate aminotransferase; ALT, blood urea nitrogen; BUN were investigated by the appropriate kit (Nanjing Jiancheng Bioengineering Institute). Additionally, major organs were further investigated using hematoxylin and eosin (H&E) by the pathology and PDX efficacy evaluation center of China Pharmaceutical University.
6.2 Acute oral toxicity assay ICR mice were divided into 4 groups (n = 6), including 17 400 mg· kg -1 , and vehicle groups for males and females, respectively.
Experimental mice were treated with 17 through oral administration once. Body weight and the heart, kidney, liver, spleen, and lung weight were recorded. Blood was collected, and the samples were analyzed by blood biochemistry assays (CRE, AST, ALT, BUN).